Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis by a SAM-Dependent Domain-of-Unknown-Function 692

Abstract

The domain of unknown function 692 (DUF692) is an emerging family of post-translational modification enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Members of this family are multinuclear iron-containing enzymes, and only two members have been functionally characterized to date: MbnB and TglH. Here, we used bioinformatics to select another member of the DUF692 family, ChrH, that is encoded in the genomes of the Chryseobacterium genus along with a partner protein ChrI. We structurally characterized the ChrH reaction product and show that the enzyme complex catalyzes an unprecedented chemical transformation that results in the formation of a macrocycle, an imidazolidinedione heterocycle, two thioaminals, and a thiomethyl group. Based on isotopic labeling studies, we propose a mechanism for the four-electron oxidation and methylation of the substrate peptide. This work identifies the first SAM-dependent reaction catalyzed by a DUF692 enzyme complex, further expanding the repertoire of remarkable reactions catalyzed by these enzymes. Based on the three currently characterized DUF692 family members, we suggest the family be called multinuclear non-heme iron dependent oxidative enzymes (MNIOs).

N–Cα Bond Cleavage Catalyzed by a Multinuclear Iron Oxygenase from a Divergent Methanobactin-like RiPP Gene Cluster

“…Furthermore, MovX catalysis proceeded without the addition of external reductant or cosubstrates, in support of earlier reports in which MNIOs extract all necessary reducing equivalents from their respective substrates. 19,22,24 However, the addition of ascorbate did increase turnover (Figure 3B), likely by reducing one of the Fe(III) ions to the catalytically active Fe(II) species. We explored three plausible mechanisms for the MovX reaction: the first involves heterolytic Lewis acid catalysis, while the second and third entail canonical oxygen activation and radical chemistry (Figure 5).…”

“…Finally, we considered H-atom abstraction from Cβ, as proposed for the three MNIOs studied thus far (Figure 5C). 19,22,24 In this case, H-atom abstraction would generate an Asn Cβ-based radical that upon oxygen rebound would give β-OH-Asn and the Fe(IV)-oxo species. Hydroxyl radical formation, mediated by the highly oxidizing Fe(IV)-oxo species, would facilitate βscission, as observed for 2-hydroxyethylphosphonate dioxygenase (HEPD) 33 and proposed for TglH, 22 giving rise to a Cαbased radical and 2-oxoacetamide.…”

“…22,23 dependent MNIO and has been shown to introduce a complex set of alterations onto its precursor including thioether macrocyclization, imidazolidinedione heterocycle formation, and thiomethylation. 24 Over 10,000 MNIOs can be seen in microbial genomes, with the vast majority uncharacterized, evidence of new chemical space that can be charted by focusing on this enzyme family. Motivated by the biosynthetic promise of MNIOs, we recently generated a sequence similarity network (SSN) 26 of DUF692 enzymes in RiPP biosynthesis, using the nearubiquitous feature of transporters in RiPP BGCs as a bioinformatic hook in a co-occurrence search.…”

Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides

“…In particular, the P450s adjacent to the emerging family of PTM enzymes termed domain of unknown function 692 (DUF692) or multinuclear nonheme iron dependent oxidative enzymes (MNIOs) are intriguing too. Members of MNIOs usually catalyze unprecedented chemical transformations in RiPP biosynthesis, and only three members have been functionally characterized . It should be noted that, apart from the P450s shown in Table S1, other P450s in this GNN may also be involved in RiPP PTMs.…”

Proteases Involved in Leader Peptide Removal during RiPP Biosynthesis

“…Most early investigations into RiPP natural products have been bioactivity-guided efforts leading to notable discoveries such as nisin, microcin B17, and darobactin. − Advances in bioinformatic technologies, such as antiSMASH, BAGEL, RODEO, RiPPer, the tools of the Enzyme Function Initiative, and many others, have enabled rapid identification of RiPP BGCs within the ever-growing genome databases. ,− Consequently, renewed efforts have focused on the discovery of novel RiPPs by genome mining (e.g., refs and − ). In addition, many studies have reported engineering of RiPP pathways to make new-to-nature structures and hybrid RiPPs. ,− A current bottleneck in such research is the identification of proteases that recognize the cognate RiPPs and remove the LP to yield the final, mature compound.…”